Dopaminergic neurons and proliferation-competent precursor cells for treating Parkinson&#39;s disease

ABSTRACT

This disclosure provides improved methods for obtaining populations of neural progenitor cells and differentiated neurons from pluripotent stem cells. The technology can be used to produce progenitors that proliferate through at least  40  doublings, while maintaining the ability to differentiate into a variety of different neural phenotypes. Cell populations have been obtained that contain a high proportion of cells staining for tyrosine hydroxylase, which is a feature of dopaminergic neurons. The neural progenitors and terminally differentiated neurons of this invention can be generated in large quantities for use in drug screening and the treatment of clinically important neurological disorders, such as Parkinson&#39;s disease.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of pending U.S. utilitypatent application Ser. No. 10/157,288, filed May 28, 2002; Ser. No.09/859,351, filed on May 16, 2001; Ser. No. 09/872,183, filed on May 31,2001; and Ser. No. 09/888,309, filed on Jun. 21, 2001. This applicationclaims priority to U.S. Provisional Patent Applications 60/205,600,filed May 17, 2000; U.S. Ser. No. 60/213,739, filed Jun. 22, 2000; and60/257,608, filed Dec. 22, 2000. This application also claims priorityto International Application PCT/US01/15861 (094/200pct), filed on May16, 2001, designating the U.S. and published on Nov. 22, 2001 as WO01/88104; and PCT/US02/19477, filed on Jun. 6, 2002 (094/300pct),designating the U.S. and published on Jan. 3, 2003 as WO 03/000868.

All the aforelisted priority applications are hereby incorporated hereinby reference in their entirety, along with International PatentPublication WO 01/51616 and WO 03/020920, with respect to the culturingand differentiation of primate pluripotent stem cells, and theproduction and use of pPS derived neural cells.

BACKGROUND

New research into the derivation and expansion of cell lines suitablefor human administration promises to usher in a brave new world medicalcare. Devastating and previously intractable disease conditions mayyield to the promise of regenerative medicine, providing that sciencecontinues to benefit from important new discoveries in the cell biologyof neurons and neural precursor cells.

Amongst the disease conditions in need of a clinical advance are thoserelating to neurological dysfunction. Near the top of the list isParkinson's disease, an idiopathic, slowly progressive, degenerativedisorder of the central nervous system, characterized by slow anddecreased movement, muscular rigidity, resting tremor, and posturalinstability. The symptoms ensue from progressive deterioration ofpigmented neurons in the substantia nigra, locus caeruleus, and otherbrain stem dopaminergic cells, causing a depletion of theneurotransmifter dopamine. Parkinson's disease is the fourth most commonneurodegenerative disease of the elderly, affecting 0.4% of those over40, and 1% of those over 65. Regardless of the age of presentation, thedisease often has devastating consequences for those afflicted.

What makes afflictions of the nervous system so difficult to manage isthe irreversibility of the damage often sustained. A central hope forthese conditions is to develop cell populations that can reconstitutethe neural network, and bring the functions of the nervous system backin line. Anecdotal evidence shows that transplantation of fetaldopaminergic neurons may reverse the chemical abnormality in Parkinson'sdisease. But there is a severe shortage of suitable tissue.

For this reason, there is a great deal of evolving interest in neuralprogenitor cells. Various types of lineage-restricted precursor cellsrenew themselves and reside in selected sites of the central nervoussystem (Kalyani et al., Biochem. Cell Biol. 6:1051, 1998). Putativeneural restricted precursors (Mayer-Proschel et al., Neuron 19:773,1997) cells express a polysialylated isoform of the neural cell adhesionmolecule (PS-NCAM). They reportedly have the capacity to generatevarious types of neurons, but not glial cells. On the other hand,putative glial restricted precursors (Rao et al., Dev. Biol. 188: 48,1997) apparently have the capacity to form glial cells but not neurons.Putative neural precursors from fetal or adult tissue are furtherillustrated in U.S. Pat. Nos. 5,852,832; 5,654,183; 5,849,553; and5,968,829; and WO 09/50526 and WO 99/01159.

Unfortunately, it has not been shown that progenitors isolated fromneural tissue have sufficient replicative capacity to produce the numberof cells necessary for human clinical therapy.

An alternative source is pluripotent cells isolated from early embryonictissue. Embryonic stem (ES) cells were first isolated from mouse embryosover 25 years ago (G. R. Martin, Proc. NatI. Acad. Sci. U.S.A. 78:7634,1981). ES cells are believed to be capable of giving rise to progeny ofvirtually any tissue type of the same species. Li, Smith et al. (Cur.Biol. 8:971, 1998) report generation of neuronal precursors from mouseES cells by lineage selection. Bjorklund et al. reported the productionof functional dopaminergic neurons from mouse ES cells (Proc. Natl.Acad. Sci. USA 19:2344, 2002).

Human ES cells were isolated much more recently (Thomson et al., Science282:114, 1998). Human ES cells require very different conditions to keepthem in an undifferentiated state, or direct them along particulardifferentiation pathways (U.S. Pat. Nos. 6,090,622 & 6,200,806;Australian Patent AU 729377, and PCT publication WO 01/51616). For thisreason, much less is known about how to prepare relatively homogeneouscell populations from human ES cells.

There is a pressing need for technology to generate more homogeneousdifferentiated cell populations from pluripotent cells of human origin.

SUMMARY

This invention provides a system for efficient production of primatecells that have differentiated from pluripotent cells into cells of theneural lineage. The precursor and terminally differentiated cells ofthis invention can be used in a number of important applications,including drug testing and therapy to restore nervous system function.

One aspect of the invention is a population of cells comprising a highproportion of cells having features characteristic of the neurallineage, such as neuronal cells, neuronal precursors, oligodendrocytes,glial cells, and neural precursors capable of giving rise to all suchcell types. The cells can be identified based on phenotypic markers,such as A2B5, NCAM, MAP-2, Nestin, β-tubulin III, and others listedlater in this disclosure, and by characteristic morphological andfunctional criteria.

Another aspect of the invention is a method of making populationscomprising neural cells from pluripotent cells, such as embryonic stemcells, embryonic germ cells, primary embryonic tissue, or stem cellsfrom fetal or adult tissue that have the capacity of differentiating (orbeing reprogrammed) into cells with a neural phenotype. The methodinvolves culturing the cells with a combination of soluble factors andenvironmental conditions that are conducive to outgrowth of neural cellswith certain desired properties. The invention includes a strategy foroptimizing differentiation protocols for differentiating pluripotentstem cells into neural cells, in which candidate factors are groupedaccording to function, and the stem cells or their progeny are culturedwith factor groups in various combinations. The groups important forproducing the desired cell type are identified, and then the individualcomponents of each group are removed one by one to determine the minimalcomposition required.

By way of illustration, pluripotent stem cells can be produced by directdifferentiation on a solid surface in the presence of one or more addedTGF-β superfamily antagonists, such as noggin and follistatin.Alternatively, pluripotent stem cells can be cultured as clusters orembryoid bodies. Enrichment for neural cells of varying degrees ofmaturity comprises culturing in a medium containing added mitogens orgrowth factors (such as EGF and FGF), concurrently or followed by addedneurotrophins (such as NT-3 or BDNF) and other factors (such as EPO) invarious optimized combinations. Lists of differentiation factors usefulin certain circumstances are listed in the general description andillustrative examples that follow. Optionally, the practitioner may alsoemploy a physical separation technique or manipulation technique thatfurther facilitates enrichment of the cells.

Mature neurons and their precursors prepared according to this inventioncan be characterized as being progeny of the cell population or anestablished cell line from which they were derived. This can bedemonstrated by showing the genome of the neural cells is essentiallythe same as that of the parent population, by some suitable techniquesuch as standard DNA fingerprinting. Alternatively, the relationship canbe established by review of records kept during derivation of the neuralcells. The characteristic that the neural cells are derived from theparent cell population is important in several respects. In particular,the undifferentiated cell population can be used for producingadditional cells with a shared genome—either a further batch of neuralcells, or another cell type that may be useful in therapy—such as apopulation that can pretolerize the patient to the histocompatibilitytype of the neural allograft.

In one embodiment of the invention, neural cells are made from humanpluripotent cells differentiated as described into neuronal precursorcells, and then passaged in culture. Using embryonic stem cells as theoriginating cell type facilitates generation of a rapidly expandingpopulation that nonetheless maintains full capacity to undergo terminaldifferentiation into functioning neurons—either when cultured withneurotrophins in the absence of mitogens, or when administered to asuitable subject. Depending on the conditions used, precursorpopulations can be generated that have the capacity to differentiateinto a high proportion of tyrosine hydroxylase positive cells. Thisphenotype is consistent with dopaminergic neurons, desirable fortreatment of Parkinson's disease.

The cells of this invention can be used for screening a compound forneural cell toxicity or modulation. A culture is prepared containing thecompound and the neural cells, and any phenotypic or metabolic change inthe cell that results from contact with the compound is determined. Thecells being tested may be dopaminergic, serotonergic, or cholinergicneurons, sensory or motor neurons, oligodendrocytes or astrocytes, orany of the neural precursor cells described in this application. It isoften commercially valuable to determine whether the compound is toxicto cells in the population. It may also be valuable to determine it thecompound changes neurotransmitter synthesis, release, or uptake by cellsin the population, or if the compound changes electrophysiologicalcharacteristics of cells in the population.

The cells of this invention can also be used for reconstituting orsupplementing the function of the nervous system in an individual, inwhich the individual is administered with an isolated cell or cellpopulation of this invention. For this purpose, the isolated cells andcell populations are formulated as a medicament for use in treatingconditions that affect the nervous system.

These and other embodiments of the invention will be apparent from thedescription that follows.

DRAWINGS

FIG. 1 shows the growth of cells bearing neural markers that werederived from human embryonic stem cells. The upper panel shows growth ofcells maintained in the presence of CNTF, bFGF, and NT3, and then sortedfor expression of NCAM. The lower panel shows growth of cells derivedfrom four different ES cell populations maintained in the presence ofEGF, bFGF, PDGF, and IGF-1, and then sorted for expression of A2B5. TheA2B5 selected population has been passaged over 7 times, and can befurther differentiated into both neuronal and glial cells.

FIG. 2 is a fluorescence micrograph showing a cell staining for tyrosinehydroxylase (TH), a marker for dopaminergic cells. Embryoid bodies madefrom human ES cells were maintained in 10 μm retinoic acid for 4 days,plated into a neural-supportive cocktail, and then passaged into mediumcontaining 10 ng/mL NT-3 and 10 ng/mL BDNF. Certain populations of thisinvention contain >1% TH-positive cells.

FIG. 3 shows response of the neural-restricted precursors to variousneurotransmitters. Panel A shows the ratio of emission data from singlecells on two different coverslips. Both cells responded to GABA,elevated potassium, acetylcholine and ATP. Panel B shows the frequencyof cells tested that responded to specific neurotransmitters. Panel Cshows the combinations of neurotransmitter responses observed.

FIG. 4 shows electrophysiology of neural-restricted precursors. Panel Ashows sodium and potassium currents observed in two cells depolarized totest potentials between −80 and 80 mV from a holding potential of −100mV. Panel B shows the inward (Na⁺) and outward (K) peak current-voltagerelationships observed. Panel C shows action potentials generated by thesame cells in response to depolarizing stimuli. These measurements showthat neural precursor cells derived from human ES cells are capable ofgenerating action potentials characteristic of neurotransmission.

FIG. 5 is a fluorescence micrograph showing neuronal cells obtained bydirect differentiation of ES cells on a solid substrate using a mixtureof differentiation factors. The three fields shown were all taken fromtreatments that comprised neurotrophins and the TNF-β superfamilyantagonists noggin and follistatin. A number of cells are seen that haveneuronal processes and stain for the neuronal marker β-tubulin-III. Theproportion of MAP-2 positive cells that were also positive for tyrosinehydroxylase (a marker for dopaminergic neurons) was as high as ˜15%.

FIG. 6 shows aspects of making neurons from hES cells by directdifferentiation. Yield of β-tubulin positive neurons is high whenundifferentiated cells are plated on laminin and cultured with the TGF-βsuperfamily antagonists noggin (N) and follistatin (F) (Panel A). Yieldwas further enhanced in the presence of stem cell factors but notmitogens (Treatment F, Panel B). Retinoic acid increased the number ofneurons produced (Panel C), but reduced the proportion of neuronsstaining positively for tyrosine hydroxylase (TH) (Panel D).

FIG. 7 shows aspects of making neurons in which differentiation wasinitiated by culturing hES to form embryoid bodies. The cells were thencultured in mitogens, subject to differential trypsinization, and thenput through multiple passages in a medium containing a cocktail ofmitogens or neurotrophic factors. When both mitogens and neurotrophinswere used, the cells could be passaged through about 40 doublings (PanelA), retaining proliferative capacity and the ability to differentiateinto mature neurons (Panel B).

FIG. 8 shows that passaging the cells in a mixture of epidermal growthfactor (EGF), basic fibroblast growth factor (FGF-2), brain-derivedneurotrophic factor (BDNF) and neurotrophin 3 (NT-3) generatedpopulations of neural precursors which upon differentiation producedcell populations that comprised ˜7% TH-positive cells, as a percentageof total cells in the population (Panel A). The cocktail used forterminal differentiation of the precursor cells can also improve theproduction of TH-positive cells (Panel B).

DETAILED DESCRIPTION

It has been discovered that when pluripotent stem cells are cultured inthe presence of selected differentiating agents, a population of cellsis derived that has a remarkably high proportion of cells withphenotypic characteristics of mature neural cells or their precursors.These cells are suitable for use in drug screening and the therapy ofconditions related to abnormalities of the nervous system.

The system encompassed by this invention is illustrated by cellpopulations obtained from an established line of human embryonic stem(hES) cells. Differentiation can be initiated by forming embryoidbodies, or by culturing the hES cells with TGF-β superfamilyantagonists.

Neurons obtained according to this invention have extended processescharacteristic of this cell type, show staining for neuron-specificmarkers like neurofilament and MAP-2, and show evidence of synapseformation, as detected by staining for synaptophysin. FIG. 3 shows thatthese cells respond to a variety of neurotransmitter substances. FIG. 4shows that these cells are capable of action potentials as measured in astandard patch-clamp system. In all these respects, the cells areapparently capable of full neurological function.

Neural precursors formed from hES cells can be passaged in culturethrough about 40 doublings, as shown in FIG. 7(A). Remarkably, evenafter multiple passages, the cells retain full capacity to differentiateinto mature neurons, as shown in FIG. 7(B). This powerful combination ofproliferative capacity and differentiation capacity has not previouslybeen available for human neural cells in culture.

Of particular interest is the capacity of this system to be adjusted tooptimize the proportion of precursors capable of generating neurons withtherapeutically important features. FIGS. 2 and 5 show neurons stainingpositively for tyrosine hydroxylase, characteristic of dopaminergicneurons. Cells of this type are particularly desirable for the treatmentof Parkinson's disease, but no other source described previously cansupply the right kind of cells with sufficient abundance. As shown inFIG. 8, passaging precursor cells in a medium containing mitogens EGFand FGF-2, and neurotrophins BDNF and NT-3 generates a proliferatingcell population capable of generating ˜7% TH-positive cells, as apercentage of total cells in the population.

Since pluripotent stem cells and some of the lineage-restrictedprecursors of this invention proliferate extensively in culture, thesystem described in this disclosure provides an unbounded supply ofneuronal and glial cells for use in research, pharmaceuticaldevelopment, and the therapeutic management of CNS abnormalities. Thepreparation and utilization of the cells of this invention isillustrated further in the description that follows.

Definitions

For the purposes of this disclosure, the terms “neural progenitor cell”or “neural precursor cell” mean a cell that can generate progeny thatare either neuronal cells (such as neuronal precursors or matureneurons) or glial cells (such as glial precursors, mature astrocytes, ormature oligodendrocytes). Typically, the cells express some of thephenotypic markers that are characteristic of the neural lineage.Typically, they do not produce progeny of other embryonic germ layerswhen cultured by themselves in vitro, unless dedifferentiated orreprogrammed in some fashion.

A “neuronal progenitor cell” or “neuronal precursor cell” is a cell thatcan generate progeny that are mature neurons. These cells may or may notalso have the capability to generate glial cells.

A “glial progenitor cell” or “glial precursor cell” is a cell that cangenerate progeny that are mature astrocytes or mature oligodendrocytes.These cells may or may not also have the capability to generate neuronalcells.

A “multipotent neural progenitor cell population” is a cell populationthat has the capability to generate both progeny that are neuronal cells(such as neuronal precursors or mature neurons), and progeny that areglial cells (such as glial precursors, mature astrocytes, or matureoligodendrocytes), and sometimes other types of cells. The term does notrequire that individual cells within the population have the capabilityof forming both types of progeny, although individual cells that aremultipotent neural progenitors may be present.

In the context of cell ontogeny, the adjective “differentiated” is arelative term. A “differentiated cell” is a cell that has progressedfurther down the developmental pathway than the cell it is beingcompared with. Thus, pluripotent embryonic stem cells can differentiateto lineage-restricted precursor cells, such as the various types ofneural progenitors listed above. These in turn can be differentiatedfurther to other types of precursor cells further down the pathway, orto an end-stage differentiated cell, such as neurons, astrocytes, oroligodendrocytes.

A “differentiation agent”, as used in this disclosure, refers to one ofa collection of compounds that are used in culture systems of thisinvention to produce differentiated cells of the neural lineage(including precursor cells and terminally differentiated cells). Nolimitation is intended as to the mode of action of the compound. Forexample, the agent may assist the differentiation process by inducing orassisting a change in phenotype, promoting growth of cells with aparticular phenotype or retarding the growth of others, or acting inconcert with other agents through unknown mechanisms.

Prototype “primate Pluripotent Stem cells” (pPS cells) are pluripotentcells derived from pre-embryonic, embryonic, or fetal tissue at any timeafter fertilization, and have the characteristic of being capable underappropriate conditions of producing progeny of several different celltypes that are derivatives of all of the three germinal layers(endoderm, mesoderm, and ectoderm), according to a standard art-acceptedtest, such as the ability to form a teratoma in 8-12 week old SCID mice.Included in the definition of pPS cells are embryonic cells of varioustypes, exemplified by human embryonic stem (hES) cells, and humanembryonic germ (hEG) cells. The pPS cells are preferably not derivedfrom a malignant source. It is desirable (but not always necessary) thatthe cells be euploid.

pPS cell cultures are described as “undifferentiated” when a substantialproportion of stem cells and their derivatives in the population displaymorphological characteristics of undifferentiated cells, clearlydistinguishing them from differentiated cells of embryo or adult origin.Undifferentiated pPS cells are easily recognized by those skilled in theart, and typically appear in the two dimensions of a microscopic view incolonies of cells with high nuclear/cytoplasmic ratios and prominentnucleoli. It is understood that colonies of undifferentiated cellswithin the population will often be surrounded by neighboring cells thatare differentiated.

“Feeder cells” or “eeders” are terms used to describe cells of one typethat are co-cultured with cells of another type, to provide anenvironment in which the cells of the second type can grow. pPS cellpopulations are said to be “essentially free” of feeder cells if thecells have been grown through at least one round after splitting inwhich fresh feeder cells are not added to support the growth of pPScells. Compositions containing less than 1%, 0.2%, 0.05%, or 0.01%feeder cells are increasingly more preferred.

The term “embryoid bodies” refers to aggregates of differentiated andundifferentiated cells that appear when pPS cells overgrow in monolayercultures, or are maintained in suspension cultures. Embryoid bodies area mixture of different cell types, typically from several germ layers,distinguishable by morphological criteria and cell markers detectable byimmunocytochemistry.

A “growth environment” is an environment in which cells of interest willproliferate, differentiate, or mature in vitro. Features of theenvironment include the medium in which the cells are cultured, anygrowth factors or differentiation-inducing factors that may be present,and a supporting structure (such as a substrate on a solid surface) ifpresent.

A cell is said to be “genetically altered”, “Iransfected”, or“genetically transformed” when a polynucleotide has been transferredinto the cell by any suitable means of artificial manipulation, or wherethe cell is a progeny of the originally altered cell that has inheritedthe polynucleotide. The polynucleotide will often comprise atranscribable sequence encoding a protein of interest, which enables thecell to express the protein at an elevated level. The genetic alterationis said to be “inheritable” if progeny of the altered cell have the samealteration.

General Techniques

For further elaboration of general techniques useful in the practice ofthis invention, the practitioner can refer to standard textbooks andreviews in cell biology, tissue culture, and embryology. Included areProperties and uses of Embryonic Stem Cells: Prospects for Applicationto Human Biology and Gene Therapy (P. D. Rathjen et al., Reprod. Fertil.Dev. 10:31, 1998); and Embryonic Stem Cells: Methods and Protocol,Kursad Turksen, ed., Humana Press, 2002.

For elaboration of nervous system abnormalities, and thecharacterization of various types of nerve cells, markers, and relatedsoluble factors, the reader is referred to CNS Regeneration: BasicScience and Clinical Advances, M. H. Tuszynski & J. H. Kordower, eds.,Academic Press, 1999. Care and feeding of neural cells is described inThe Neuron: Cell and Molecular Biology, 3^(rd) Edition, I. B. Levitan &L. K. Kaczmarek, Oxford U. Press, 2001; and The Neuron in TissueCulture, L. W. Haynes Ed., John Wiley & Son Ltd, 1999.

Sources of Stem Cells

This invention can be practiced with pluripotent stem cells of varioustypes, particularly stem cells derived from embryonic tissue and havethe characteristic of being capable of producing progeny of all of thethree germinal layers, as described above.

Exemplary are embryonic stem cells and embryonic germ cells used asexisting cell lines or established from primary embryonic tissue of aprimate species, including humans. This invention can also be practicedusing pluripotent cells obtained from primary embryonic tissue, withoutfirst establishing an undifferentiated cell line.

Embryonic Stem Cells

Embryonic stem cells can be isolated from blastocysts of primate species(U.S. Pat. No. 5,843,780; Thomson et al., Proc. NatI. Acad. Sci. USA92:7844, 1995). Human embryonic stem (hES) cells can be prepared fromhuman blastocyst cells using the techniques described by Thomson et al.(U.S. Pat. No. 6,200,806; Science 282:1145, 1998; Curr. Top. Dev. Biol.38:133 ff., 1998) and Reubinoff et al, Nature Biotech. 18:399, 2000.Equivalent cell types to hES cells include their pluripotentderivatives, such as primitive ectoderm-like (EPL) cells, outlined in WO01/51610 (Bresagen).

hES cells can be obtained from human preimplantation embryos (Thomson etal., Science 282:1145, 1998). Alternatively, in vitro fertilized (IVF)embryos can be used, or one-cell human embryos can be expanded to theblastocyst stage (Bongso et al., Hum Reprod 4: 706, 1989). Embryos arecultured to the blastocyst stage , the zona pellucida is removed, andthe inner cell masses are isolated (for example, by immunosurgery usingrabbit anti-human spleen cell antiserum). The intact inner cell mass isplated on mEF feeder layers, and after 9 to 15 days, inner cell massderived outgrowths are dissociated into clumps. Growing colonies havingundifferentiated morphology are dissociated into clumps, and replated.ES-like morphology is characterized as compact colonies with apparentlyhigh nucleus to cytoplasm ratio and prominent nucleoli. Resulting EScells are then routinely split every 1-2 weeks. Clump sizes of about 50to 100 cells are optimal.

Propagation of pPS Cells in an Undifferentiated State

pPS cells can be propagated continuously in culture, using cultureconditions that promote proliferation while inhibiting differentiation.Exemplary serum-containing ES medium is made with 80% DMEM (such asKnock-Out DMEM, Gibco), 20% of either defined fetal bovine serum (FBS,Hyclone) or serum replacement (U.S. 2002/0076747 A1, Life TechnologiesInc.), 1% non-essential amino acids, 1 mM L-glutamine, and 0.1 mMβ-mercaptoethanol.

Traditionally, ES cells are cultured on a layer of feeder cells,typically fibroblasts derived from embryonic or fetal tissue (Thomson etal., Science 282:1145, 1998). Scientists at Geron have discovered thatpPS cells can be maintained in an undifferentiated state even withoutfeeder cells. The environment for feeder-free cultures includes asuitable culture substrate, particularly an extracellular matrix such asMatrigel® or laminin. The pPS cells are plated at >15,000 cells cm⁻²(optimally 90,000 cm⁻² to 170,000 cm⁻²). Typically, enzymatic digestionis halted before cells become completely dispersed (say, ˜5 min withcollagenase IV). Clumps of ˜10 to 2,000 cells are then plated directlyonto the substrate without further dispersal. Alternatively, the cellscan be harvested without enzymes before the plate reaches confluence byincubating ˜5 min in a solution of 0.5 mM EDTA in PBS. After washingfrom the culture vessel, the cells are plated into a new culture withoutfurther dispersal. In a further illustration, confluent hES cellscultured in the absence of feeders are removed from the plates byincubating with a solution of 0.05% (wt/vol) trypsin (Gibco) and 0.053mM EDTA for 5-15 min at 37° C. The remaining cells in the plate areremoved and the cells are triturated into a suspension comprising singlecells and small clusters, and then plated at densities of 50,000-200,000cells cm⁻² to promote survival and limit differentiation.

Feeder-free cultures are supported by a nutrient medium containingfactors that promote proliferation of the cells without differentiation(WO 99/20741). Such factors may be introduced into the medium byculturing the medium with cells secreting such factors, such asirradiated (˜4,000 rad) primary mouse embryonic fibroblasts, telomerizedmouse fibroblasts, or fibroblast-like cells derived from pPS cells (U.S.Pat. No. 6,642,048). Medium can be conditioned by plating the feeders ina serum free medium such as KO DMEM supplemented with 20% serumreplacement and 4 ng/mL bFGF. Medium that has been conditioned for 1-2days is supplemented with further bFGF, and used to support pPS cellculture for 1-2 days (WO 01/51616; Xu et al., Nat. Biotechnol. 19:971,2001).

Alternatively, fresh or non-conditioned medium can be used, which hasbeen supplemented with added factors (like a fibroblast growth factor orforskolin) that promote proliferation of the cells in anundifferentiated form. Exemplary is a base medium like X-VIVO™ 10(Biowhittaker) or QBSF™-60 (Quality Biological Inc.), supplemented withbFGF at 40-80 ng/mL, and optionally containing stem cell factor (15ng/mL), or Flt3 ligand (75 ng/mL). These medium formulations have theadvantage of supporting cell growth at 2-3 times the rate in othersystems.

Under the microscope, ES cells appear with high nuclear/cytoplasmicratios, prominent nucleoli, and compact colony formation with poorlydiscernable cell junctions. Primate ES cells typically express thestage-specific embryonic antigens (SSEA) 3 and 4, and markers detectableusing antibodies designated Tra-1-60 and Tra-1-81. Undifferentiated hEScells also typically express the transcription factor Oct-3/4, Cripto,gastrin-releasing peptide (GRP) receptor, podocalyxin-like protein(PODXL), and human telomerase reverse transcriptase (hTERT) (U.S.2003/0224411 A1), as detected by RT-PCR.

Materials and Procedures for Preparing Neural Precursors and TerminallyDifferentiated Cells

The neural progenitors and mature neurons of this invention can be madeby differentiating stem cells using a suitable differentiation paradigm.

Typically, differentiation protocols are conducted in a cultureenvironment comprising a suitable substrate, and a nutrient medium towhich the differentiation agents are added. Suitable substrates includesolid surfaces coated with a positive charge, exemplified bypoly-L-lysine and polyornithine. Substrates can be coated withextracellular matrix components, exemplified by fibronectin and laminin.Other permissive extracellular matrixes include Matrigel® (extracellularmatrix from Engelbreth-Holm-Swarm tumor cells). Also suitable arecombination substrates, such as poly-L-lysine combined with fibronectin,laminin, or both.

Neural lineage cells of this invention are cultured in a medium thatsupports the proliferation or survival of the desired cell type. It isoften desirable to use a defined medium that supplies nutrients as freeamino acids rather than serum. It is also beneficial to supplement themedium with additives developed for sustained cultures of neural cells.Exemplary are N2 and B27 additives, available commercially from Gibco.

Advancing cells along the neural differentiation pathway is promoted byincluding in the culture medium a cocktail of differentiation agentsthat enhances outgrowth of the desired cell type. This may involvedirecting the cells or their progeny to adopt phenotypic features of thedifferentiated cell type, promoting the growth of cells with the desiredphenotype, or inhibiting growth of other cell types. It is usually notnecessary to understand the mode of action of the agents in order topractice the invention.

Suitable differentiation agents include growth factors of various kinds,such as epidermal growth factor (EGF), transforming growth factor a(TGF-α), any type of fibroblast growth factor (exemplified by FGF-4,FGF-8, and basic fibroblast growth factor=bFGF), platelet-derived growthfactor (PDGF), insulin-like growth factor (IGF-1 and others), highconcentrations of insulin, sonic hedgehog, members of the neurotrophinfamily (such as nerve growth factor=NGF, neurotrophin 3=NT-3,brain-derived neurotrophic factor=BDNF), bone morphogenic proteins(especially BMP-2 & BMP-4), retinoic acid (RA) and ligands to receptorsthat complex with gp130 (such as LIF, CNTF, and IL-6). Also suitable arealternative ligands and antibodies that bind to the respectivecell-surface receptors for the aforementioned factors. Typically, aplurality of differentiation agents is used, which may comprise 2, 3, 4,or more of the agents listed above or in the examples below.

In one differentiation method, pPS cells are plated directly onto asuitable substrate, such as an adherent glass or plastic surface, suchas coverslips coated with poly-lysine, with or without a neuron-friendlymatrix protein such as fibronectin or laminin. The cells are thencultured in a suitable nutrient medium that is adapted to promotedifferentiation towards neural cells. This is referred to as the “directdifferentiation” method, which is further illustrated in InternationalPatent Publication WO 01/51616, and priority U.S. patent applicationSer. No. 09/888,309. TGF-β superfamily antagonists such as noggin andfollistatin are especially useful in directing neural differentiationand enhancing the proportion of cells bearing phenotypic features ofneural cells obtained by direct differentiation (Example 9).

In another differentiation method, pPS cells are firstpre-differentiated into a heterogeneous cell population by forming cellclusters. In an exemplary variation, embryoid bodies are formed from thepPS cells by culturing them in suspension. Optionally, one or more ofthe differentiation agents listed earlier (such as retinoic acid) can beincluded in the medium to promote differentiation within the embryoidbody. After the embryoid bodies have reached sufficient size or maturity(typically 3-4 days), they are plated onto the substrate of thedifferentiation culture. The embryoid bodies can be plated directly ontothe substrate without dispersing the cells. This allows neural cellprecursors to migrate out of the embryoid bodies and on to theextracellular matrix. In some procedures, the cells are first culturedin a mitogen cocktail, such as EGF, bFGF, PDGF, and IGF-1, and thenpassaged in a combination of mitogens and neurotrophins to select outneural progenitor cells.

This invention includes a strategy for identifying factor combinationseffective for generating particular neural phenotypes. Various factorsknown or suspected to enhance neural differentiation or growth arecategorized into various functional classes, based on known effects onneural cells from other tissues or species, known receptor bindingactivities, structural homology with other factors of known function, orother appropriate criteria. Factors within each class are pooled at asuitable working concentration. Cells are then cultured with each of thefactor classes together, in various combinations, and the factors areassessed on the ability to promote growth of precursor cells or matureneurons of the desired type. Essential factor classes are identifiedwhen their absence causes the mixture to lose its ability to promote thedesired phenotype. Once essential classes are identified and others areeliminated, then each of the classes is dissected by removing singlecomponents until the minimal cocktail is identified. The implementationof this strategy is illustrated in Example 9.

If desired, the differentiated cells can be sorted to enrich for certainpopulations. For example, the cells can be contacted with an antibody orligand that binds to a marker characteristic of neural cells (such asNCAM), followed by separation of the specifically recognized cells usinga suitable immunological technique, such as solid phase adsorption orfluorescence-activated cell sorting. Also suitable are differentialplating or harvesting techniques, in which adherence or releasability ofthe desired cell type is used to separate it from other cells in aheterogeneous population.

It has been discovered that neural precursor phenotype can be passagedin proliferating culture using a combination of mitogens (such as bFGFand EGF), plus one or more neurotrophins (such as BDNF, NT-3, or both).This is illustrated in Examples 6, 9 and 10. The cells can be passagedfor up to 40 doublings according to this method (FIG. 7), whileretaining both an ability to proliferate and an ability to make matureneurons.

It is hypothesized that committed progenitor cells will have particularvalue in human therapy, because they are more resilient to manipulation,and will retain a greater ability to migrate to the target tissue andintegrate in a functionally compatible fashion. Progenitor cells can begrown either on a solid surface as illustrated in Example 10, or insuspension culture, where they tend to form clusters or sphericalstructures. By way of illustration, neural progenitors are harvestedusing trypsin when nearly confluent. They are then seeded at about halfdensity in nonadherent wells, and cultured in supplemented mediumcontaining 10 ng/mL of BDNF, NT-3, EGF, and bFGF, changed about 3 timesper week.

Judicious selection of other components of the culture medium duringderivation or maintenance of the neural progenitor cells can influencethe range and character of mature cells that they can generate. Asillustrated in Example 9, including retinoic acid in the medium duringdirect differentiation of neural progenitors increases the proportion ofMAP-2 cells produced upon terminal differentiation—but decreases theproportion of cells positive for tyrosine hydroxylase (TH), whichcorrelates with dopaminergic neurons. On the other hand, it has beendiscovered that including erythropoietin (EPO) or agents that increasecyclic AMP levels in the culture medium during neural progenitorformation enhances the capacity for forming TH positive neurons. As analternative, cells can be cultured with certain antibodies or agoniststhat activate the EPO pathway, or the cells can be cultured under mildlyhypoxic conditions (low O₂ levels, say 3-6%). Use of EPO to enhanceformation of the dopaminergic phenotype is illustrated in Example 7.

Neural precursor cells prepared according to any of these procedures canbe further differentiated to mature neurons. Fully differentiated cellsare desirable for various applications of this invention, such as the invitro assessment and screening of various compounds for their effect onneural tissue. It is also useful to make fully differentiated cells tocharacterize the functional capabilities of neural progenitors fromwhich they came.

Differentiated neurons can be formed by culturing precursor cells with amaturation factor, such as forskolin (or other compound that elevatesintracellular cAMP levels such as cholera toxin, isobutylmethylxanthine,dibutyladenosine cyclic monophosphate), c-kit ligand, retinoic acid, orany factor or combination of factors from the family of neurotrophins.Particularly effective are neurotrophin-3 (NT-3) in combination withbrain-derived neurotrophic factor (BDNF). Other candidates are GDNF,BMP-2, and BMP-4. Alternatively or in addition, maturation can beenhanced by withdrawing some or all of the factors that promote neuralprecursor proliferation, such as EGF, FGF, or other mitogens previouslyused to maintain the culture.

Possible Further Adaptations

Many of the neural cell precursor populations of this invention have asubstantial proliferation capacity. If desired, the replication capacitycan be further enhanced by increasing the level of telomerase reversetranscriptase (TERT) in the cell, either by increasing transcriptionfrom the endogenous gene, or by introducing a transgene. Particularlysuitable is the catalytic component of human telomerase (hTERT),provided in International Patent Application WO 98/14592. Transfectionand expression of telomerase in human cells is described in Bodnar etal., Science 279:349, 1998 and Jiang et al., Nat. Genet. 21:111, 1999.Genetically altered cells can be assessed for hTERT expression byRT-PCR, telomerase activity (TRAP assay), immunocytochemical stainingfor hTERT, or replicative capacity, according to standard methods.

For use in therapeutic and other applications, it is often desirablethat populations of precursor or mature neurological cells besubstantially free of undifferentiated pPS cells. One way of depletingundifferentiated stem cells from the population is to transfect themwith a vector in which an effector gene under control of a promoter thatcauses preferential expression in undifferentiated cells. Suitablepromoters include the TERT promoter and the OCT-4 promoter. The effectorgene may be directly lytic to the cell (encoding, for example, a toxinor a mediator of apoptosis). Alternatively, the effector gene may renderthe cell susceptible to toxic effects of an external agent, such as anantibody or a prodrug. Exemplary is a herpes simplex thymidine kinase(tk) gene, which causes cells in which it is expressed to be susceptibleto ganciclovir. Suitable pTERT-tk constructs are provided inInternational Patent Publication WO 98/14593 (Morin et al.).

Characteristics of Neural Precursors and Terminally Differentiated Cells

Cells can be characterized according to a number of phenotypic criteria,such as morphological features, detection or quantitation of expressedcell markers, enzymatic activity, or neurotransmitters and theirreceptors, and electrophysiological function.

Certain cells embodied in this invention have morphological featurescharacteristic of neuronal cells or glial cells. The features arereadily appreciated by those skilled in evaluating the presence of suchcells. For example, characteristic of neurons are small cell bodies, andmultiple processes reminiscent of axons and dendrites. Cells of thisinvention can also be characterized according to whether they expressphenotypic markers characteristic of neural cells of various kinds.

Markers of interest include but are not limited to β-tubulin III,microtubule-associated protein 2 (MAP-2), or neurofilament,characteristic of neurons; glial fibrillary acidic protein (GFAP),present in astrocytes; galactocerebroside (GaIC) or myelin basic protein(MBP), characteristic of oligodendrocytes; Oct-4, characteristic ofundifferentiated hES cells; and Nestin, characteristic of neuralprecursors and other cells. Both A2B5 (a glycolipid) and polysialylatedNeural Cell Adhesion Molecule (abbreviated NCAM) have already beendescribed. While A2B5 and NCAM are instructive markers when studyingneural lineage cells, it should be appreciated that these markers cansometimes be displayed on other cell types, such as liver or musclecells. β-Tubulin III was previously thought to be specific for neuralcells, but it has been discovered that a subpopulation of hES cells isalso β-tubulin lIl positive. MAP-2 is a more stringent marker for fullydifferentiated neurons of various types. Certain cell populationsprepared according to this invention comprise at least 30%, 50%, 75%,90% or more that test positive for these markers, either alone or invarious combinations.

Tissue-specific markers listed in this disclosure and known in the artcan be detected using any suitable immunological technique—such as flowimmunocytochemistry for cell-surface markers, immunohistochemistry (forexample, of fixed cells or tissue sections) for intracellular orcell-surface markers, Western blot analysis of cellular extracts, andenzyme-linked immunoassay, for cellular extracts or products secretedinto the medium. Expression of an antigen by a cell is said to be“antibody-detectable” if a significantly detectable amount of antibodywill bind to the antigen in a standard immunocytochemistry or flowcytometry assay, optionally after fixation of the cells, and optionallyusing a labeled secondary antibody or other conjugate (such as abiotin-avidin conjugate) to amplify labeling.

The expression of tissue-specific gene products can also be detected atthe mRNA level by Northern blot analysis, dot-blot hybridizationanalysis, or by reverse transcriptase initiated polymerase chainreaction (RT-PCR) using sequence-specific primers in standardamplification methods. See U.S. Pat. No. 5,843,780 for further details.Sequence data for the particular markers listed in this disclosure canbe obtained from public databases such as GenBank (URLwww.ncbi.nlm.nih.gov:80/entrez). Expression at the mRNA level is said tobe “detectable” according to one of the assays described in thisdisclosure if the performance of the assay on cell samples according tostandard procedures in a typical controlled experiment results inclearly discernable hybridization or amplification product. Expressionof tissue-specific markers as detected at the protein or mRNA level isconsidered positive if the level is at least 2-fold, and preferably morethan 10- or 50-fold above that of a control cell, such as anundifferentiated pPS cell, a fibroblast, or other unrelated cell type.

Also characteristic of neural cells, particularly terminallydifferentiated cells, are receptors and enzymes involved in thebiosynthesis, release, and reuptake of neurotransmitters, and ionchannels involved in the depolarization and repolarization events thatrelate to synaptic transmission. Evidence of synapse formation can beobtained by staining for synaptophysin. Evidence for receptivity tocertain neurotransmifters can be obtained by detecting receptors forγ-amino butyric acid (GABA), glutamate, dopamine,3,4-dihydroxyphenylalanine (DOPA), noradrenaline, acetylcholine, andserotonin.

Differentiation of particular neural precursor cell populations of thisinvention (for example, using NT-3 and BDNF) can generate cellpopulations that are at least 20%, 30%, or 40% MAP-2 positive. Asubstantial proportion, say 5%, 10%, 25%, or more of the NCAM or MAP-2positive cells (on a cell count basis) will be capable of synthesizing aneurotransmitter, such as acetylcholine, glycine, glutamate,norepinephrine, serotonin, or GABA. Certain populations of the inventioncontain NCAM or MAP-2 positive cells that have 1%, 5%, 10% or more thatare positive for tyrosine hydroxylase (TH), measured byimmunocytochemistry or mRNA expression—either as a percentage of NCAM orMAP-2 positive cells, or all cells present in the population. TH isgenerally considered in the art to be a marker for dopamine synthesizingcells.

To elucidate further mature neurons present in a differentiatedpopulation, the cells can be tested according to functional criteria.For example, calcium flux can be measured by any standard technique, inresponse to a neurotransmifter, or other environmental condition knownto affect neurons in vivo. First, neuron-like cells in the populationare identified by morphological criteria, or by a marker such as NCAM.The neurotransmifter or condition is then applied to the cell, and theresponse is monitored (Example 6). The cells can also be subjected tostandard patch-clamp techniques, to determine whether there is evidencefor an action potential, and what the lag time is between appliedpotential and response.

Where derived from an established line of pPS cells, the cellpopulations and isolated cells of this invention can be characterized ashaving the same genome as the line from which they are derived. Thismeans that the chromosomal DNA will be over 90% identical between thepPS cells and the neural cells, which can be inferred if the neuralcells are obtained from the undifferentiated line through the course ofnormal mitotic division. Neural cells that have been treated byrecombinant methods to introduce a transgene (such as TERT) or knock outan endogenous gene are still considered to have the same genome as theline from which they are derived, since all non-manipulated geneticelements are preserved.

Use of Neural Precursors and Terminally Differentiated Cells

This invention provides a method to produce large numbers of neuralprecursor cells and mature neuronal and glial cells. These cellpopulations can be used for important research, development, andcommercial purposes.

The cells of this invention can be used to prepare a cDNA libraryrelatively uncontaminated with cDNA preferentially expressed in cellsfrom other lineages. For example, multipotent neural progenitor cellsare collected by centrifugation at 1000 rpm for 5 min, and then mRNA isprepared, reverse transcribed, and optionally subtracted with cDNA frommature neurons, astrocytes, or oligodendrocytes, or undifferentiatedastrocytes. Expression patterns of neurons can be compared with othercell types by microarray analysis, reviewed generally by Fritz et alScience 288:316, 2000; “Microarray Biochip Technology”, L Shi,www.Gene-Chips.com.

The differentiated cells of this invention can also be used to prepareantibodies that are specific for markers of multipotent neuralprogenitors, cells committed to the neuronal or glial cell lineage, andmature neurons, astrocytes, and oligodendrocytes. Polyclonal antibodiescan be prepared by injecting a vertebrate animal with cells of thisinvention in an immunogenic form. Production of monoclonal antibodies isdescribed in such standard references as Harrow & Lane (1988), U.S. Pat.Nos. 4,491,632, 4,472,500 and 4,444,887, and Methods in Enzymology 73B:3(1981).

Applications of commercial interest include the use of cells to screensmall molecule drugs, and the preparation of pharmaceutical compositionscomprising neurons for clinical therapy.

Drug Screening

Neural precursor cells of this invention can be used to screen forfactors (such as solvents, small molecule drugs, peptides,polynucleotides) or environmental conditions (such as culture conditionsor manipulation) that affect the characteristics of neural precursorcells and their various progeny.

In some applications, pPS cells (undifferentiated or differentiated) areused to screen factors that promote maturation into neural cells, orpromote proliferation and maintenance of such cells in long-termculture. For example, candidate maturation factors or growth factors aretested by adding them to cells in different wells, and then determiningany phenotypic change that results, according to desirable criteria forfurther culture and use of the cells.

Other screening applications of this invention relate to the testing ofpharmaceutical compounds for their effect on neural tissue or nervetransmission. Screening may be done either because the compound isdesigned to have a pharmacological effect on neural cells, or because acompound designed to have effects elsewhere may have unintended sideeffects on the nervous system. The screening can be conducted using anyof the neural precursor cells or terminally differentiated cells of theinvention, such as dopaminergic, serotonergic, cholinergic, sensory, andmotor neurons, oligodendrocytes, and astrocytes.

The reader is referred generally to the standard textbook “In vitroMethods in Pharmaceutical Research”, Academic Press, 1997, and U.S. Pat.No. 5,030,015. Assessment of the activity of candidate pharmaceuticalcompounds generally involves combining the differentiated cells of thisinvention with the candidate compound, either alone or in combinationwith other drugs. The investigator determines any change in themorphology, marker phenotype, or functional activity of the cells thatis attributable to the compound (compared with untreated cells or cellstreated with an inert compound), and then correlates the effect of thecompound with the observed change.

Cytotoxicity can be determined in the first instance by the effect oncell viability, survival, morphology, and the expression of certainmarkers and receptors. Effects of a drug on chromosomal DNA can bedetermined by measuring DNA synthesis or repair. [³H]-thymidine or BrdUincorporation, especially at unscheduled times in the cell cycle, orabove the level required for cell replication, is consistent with a drugeffect. Unwanted effects can also include unusual rates of sisterchromatid exchange, determined by metaphase spread. The reader isreferred to A. Vickers (pp 375-410 in “In vitro Methods inPharmaceutical Research,” Academic Press, 1997) for further elaboration.

Effect of cell function can be assessed using any standard assay toobserve phenotype or activity of neural cells, such as receptor binding,neurotransmitter synthesis, release or uptake, electrophysiology, andthe growing of neuronal processes or myelin sheaths—either in cellculture or in an appropriate model. For example, the ability of drugs toalter synaptic contact and plasticity can be measured in culture byimmunocytochemical staining for synapsin or synaptophysin.Electrophysiology can be assessed by measuring measure IPSPs and EPSPs(inhibitory and excitatory postsynaptic potentials). Alternatively,using a two electrode system, one cell is stimulated, and the responseof a second cell in the system is evaluated. The behavior of the systemin the presence of the candidate drug is compared with the behavior inthe absence of the drug, and correlated with an ability of the drug toaffect synaptic contact or cell plasticity.

Therapeutic Use

This invention also provides for the use of neural precursor cells torestore a degree of central nervous system (CNS) function to a subjectneeding such therapy, perhaps due to an inborn error in function, theeffect of a disease condition, or the result of an injury.

To determine the suitability of neural precursor cells for therapeuticadministration, the cells can first be tested in a suitable animalmodel. At one level, cells are assessed for their ability to survive andmaintain their phenotype in vivo. Neural precursor cells areadministered to immunodeficient animals (such as nude mice, or animalsrendered immunodeficient chemically or by irradiation) at an observablesite, such as in the cerebral cavity or in the spinal cord. Tissues areharvested after a period of a few days to several weeks or more, andassessed as to whether pPS derived cells are still present.

This can be performed by administering cells that express a detectablelabel (such as green fluorescent protein, or β-galactosidase); that havebeen prelabeled (for example, with BrdU or [³H]thymidine), or bysubsequent detection of a constitutive cell marker (for example, usinghuman-specific antibody). Where neural precursor cells are being testedin a rodent model, the presence and phenotype of the administered cellscan be assessed by immunohistochemistry or ELISA using human-specificantibody, or by RT-PCR analysis using primers and hybridizationconditions that cause amplification to be specific for humanpolynucleotide sequences. Suitable markers for assessing gene expressionat the mRNA or protein level are provided elsewhere in this disclosure.

Various animal models for testing restoration of nervous system functionare described in CNS Regeneration: Basic Science and Clinical Advances,M. H. Tuszynski & J. H. Kordower, eds., Academic Press, 1999.Parkinson's disease can be modeled in rats by surgically inducingnigrostriatal lesions, thereby obstructing a major dopamine pathway inthe brain. Another standard animal model is chemical lesioning ofdopaminergic neurons in the substantia nigra of mice or non-humanprimates with MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine).Illustrations are provided in Furns et al., Proc. Natl. Acad. Sci. USA80:4546, 1983; Freed et al., Appl. Neurophysiol. 47:16, 1984; andBjorklund et al., Proc. Natl. Acad. Sci. USA 19:2344, 2002.

Differentiated cells of this invention can also be used for tissuereconstitution or regeneration in a human patient in need thereof. Thecells are administered in a manner that permits them to graft or migrateto the intended tissue site and reconstitute or regenerate thefunctionally deficient area.

By way of illustration, neural stem cells are transplanted directly intoparenchymal or intrathecal sites of the central nervous system,according to the disease being treated. Grafts are done using singlecell suspension or small aggregates at a density of 25,000-500,000 cellsper pL. (U.S. Pat. No. 5,968,829). Certain neural progenitor cellsembodied in this invention are designed for treatment of acute orchronic damage to the nervous system. For example, excitotoxicity hasbeen implicated in a variety of conditions including epilepsy, stroke,ischemia, and Alzheimer's disease. Dopaminergic neurons may beformulated for treating Parkinson's disease, GABAergic neurons forHuntington's disease, and motor neurons for spinal cord injury oramyotrophic lateral sclerosis (ALS).

COMMERCIAL EMBODIMENTS

As stated in the summary, this disclosure provides a system forefficient production of primate cells that have differentiated frompluripotent cells into cells of the neural lineage. The skilled readerwill appreciate that both research and commercial use of this system canentail possession of the pPS cells as a renewable cell bank, and theneural lineage cells obtained from them according to the differentiationmethods described—as is illustrated in the examples that follow.

Accordingly, this invention embodies any set or combination of cells orreagents that exist at any time during manufacture, distribution, or useof the pPS derived neural cells, as described in this disclosure. Suchembodiments comprise any combination of two or more cell populationsand/or reagents described in this disclosure, exemplified but notlimited to a type of differentiated pPS-derived cell (glial cells,oligodendrocytes, their precursors and subtypes, and so on), incombination with undifferentiated pPS cells or other differentiated celltypes, sometimes sharing the same genome. Other embodiments comprise thepPS cell line in combination with the factor(s) effective todifferentiate them into useful types of neural lineage cells (such asthe neurotrophic factors, mitogens, and TGF-β superfamily agonistslisted earlier). Each cell type or reagent in the set may be packagedtogether, or in separate containers in the same facility, or atdifferent locations, at the same or different times, under control ofthe same entity or different entities sharing a business relationship.

For general principles in medicinal formulation of cell compositions,the reader is referred to Cell Therapy: Stem Cell Transplantation, GeneTherapy, and Cellular Immunotherapy, by G. Morstyn & W. Sheridan eds,Cambridge University Press, 1996; and Cell Transplantation forNeurological Disorders, T. B. Freeman et al. eds., Humana Press 1998.The composition may optionally be packaged in a suitable container withwritten instructions for a desired purpose, such as the reconstitutionof CNS function to improve some neurological abnormality, such asParkinson's Disease.

The Following Examples are Provided as Further Non-LimitingIllustrations of Particular Embodiments of the Invention EXAMPLESExample 1 NCAM-Positive Cells

This experiment focused on determining whether the human embryonic stemcells (hES) could undergo directed differentiation to NCAM-positiveprogenitor cells.

hES cells were harvested either from cultures supported by embryonicfibroblasts, or from feeder-free cultures, as described previously (AU729377; WO 01/51616). Embryoid bodies were produced as follows.Confluent monolayer cultures of hES cells were harvested by incubatingin 1 mg/mL collagenase for 5-20 min, following which the cells arescraped from the plate. The cells were then dissociated into clustersand plated in non-adherent cell culture plates (Costar) in a mediumcomposed of 80% KO (“knockout”) DMEM (Gibco) and 20%non-heat-inactivated FBS (Hyclone), supplemented with 1% non-essentialamino acids, 1 mM glutamine, 0.1 mM β-mercaptoethanol. The cells areseeded at a 1:1 or 1:2 ratio in 2 mL medium per well (6 well plate).

After 4-8 days in suspension, the EBs were plated intact in DMEM/F12medium containing B27 supplement (Gibco), N2 supplement (Gibco), and 25ng/mL human bFGF. Wells were coated with fibronectin (Sigma) at a finalconcentration of 20 μg/mL in PBS. After culturing for about 2-3 days,NCAM-positive cells and A2B5-positive cells were identified byimmunostaining.

Magnetic bead sorting and immunopanning were both successful inenriching NCAM-positive cells. The starting population of cellstypically contained 25-72% NCAM-positive cells. After immuno-isolation,the NCAM-positive proportion was enriched to 43-72%. Results are shownin Table 1. TABLE 1 Differentiation and Sorting Conditions for NCAMpositive Cells Factors Cells staining used positively for NCAM hES inDiffer- Neg- Cell Line used entiation Before Positive ative forDifferentiation Culture Type of Sort sort sort sort H13 p28 C F N beadsort 33 92 41 H13 p28 C F N panning 25 n/a n/a H9 p32 C F N panning 6472 51 H1 p32 C F N bead sort 27 77  9 H9 p19 C F N bead sort 58 76 32 H9p31 545.184 C F N bead sort 50 91 67 H1 p40 545.185 C F N bead sort A 6589 31 H1 p40 545.185 C F N bead sort B 63 81 33 H7NG p28/4 C F N beadsort A 53 92 45 545.187 H7NG p28/4 C F N bead sort B 72 87 50 545.187 H1p39 545.189 C F N I P bead sort 16 43  6 H7 p32 667.004 C F N I P beadsort 25 73 10 H1 p43 667.010 C F N I P bead sort 47 86 31 H1 p44 667.012C F N I P bead sort 52 89 34 H1 p46 667.020 EPFI bead sort 60 23  8 H1p47 667.031 EPFI— bead sort 53 91 27 EPFI H1 p47 667.033 C F N - F beadsort 41 76 24 H9 p40MG E P F I bead sort 55 80 25 667.038Factor abbreviations:C—ciliary neurotrophic factor (CNTF)F—basic fibroblast growth factor (bFGF)N—neurotrophin 3 (NT3)I—insulin-like growth factor (IGF-1)P—platelet-derived growth factor (PDGF)T—thyroid hormone T₃Ra—retinoic acidFk—Forskolin

In the first 10 experiments shown, NCAM positive cells retrieved fromthe sort were plated on poly-L-lusine/laminin in DMEM/F12 with N2 andB27 supplements and 2 mg/mL BSA, 10 ng/mL human CNTF, 10 ng/mL humanbFGF and 1 to 10 ng/mL human NT-3. In subsequent experiments, cells weremaintained in DMEM/F12 with N2 and B27 supplements and 10 ng/mL EGF, 10ng/mL bFGF, 1 ng/mL PDGF-M, and 1 ng/mL IGF-1.

FIG. 1 (Upper Panel) shows the growth curves for the NCAM positivecells. The cells studied in this experiment were prepared by formingembryoid bodies in 20% FBS for 4 days in suspension, then plating onto afibronectin matrix in DMEM/F12 with N2 and B27 supplements and 25 ng/mLbFGF for 2-3 days. The cells were then positively sorted for NCAMexpression, and maintained in a medium containing CNTF, bFGF, and NT3.The sorted cells did not show increased survival relative to theunsorted population. It was found that some of the NCAM positive cellsalso express β-tubulin III, indicating that these cells have thecapacity to form neurons. They also had morphology characteristic ofneuronal cells. There were also A2B5 positive cells within thispopulation, which may represent glial progenitor cells. However, veryfew cells were positive for GFAP, a marker for astrocytes. Although thiscell population proliferated in culture, the proportion of NCAM positivecells (and the capacity to form neurons) diminished after severalpassages.

Example 2 A2B5-Positive Cells

Cells in this experiment were immunoselected for the surface markerA2B5. hES cells were induced to form EBs in 20% FBS. After 4 days insuspension, the EBs were plated onto fibronectin in DMEM/F12 with N2 andB27 supplemented with 10 ng/mL human EGF, 10 ng/mL human bFGF, 1 ng/mLhuman IGF-I, and 1 ng/mL human PDGF-AA. After 2-3 days in theseconditions, 25-66% of the cells express A2B5. This population isenriched by magnetic bead sorting to 48-93% purity (Table 2). TABLE 2Differentiation and Sorting Conditions for A2B5-positive Cells Cellsstaining hES Factors used in positively for NCAM Cell Line used forDifferentiation Before Positive Negative Differentiation Culture Type ofSort sort sort sort H7 p32 667.004 C F N I P bead sort 25 77 10 H1 p43667.010 C F N I P bead sort 62 n/a 50 H1 p44 667.012 C F N I P bead sort56 89 32 H1 p46 667.020 E P F I bead sort 27 48  2 H1 p47 667.032 E P FI bead sort 57 93 30 H9 p40MG 667.038 E P F I bead sort 66 93 41 H9 p42667.041 E P F I bead sort 27 70  6Factor abbreviations:C—ciliary neurotrophic factor (CNTF)F—basic fibroblast growth factor (bFGF)N—neurotrophin 3 (NT3)I—insulin-like growth factor (IGF-1)P—platelet-derived growth factor (PDGF)T—thyroid hormone T₃Ra—retinoic acidFk—Forskolin

FIG. 1 (Lower Panel) shows the growth curves for the sortedA2B5-positive cells. Four different hES cell populations were used forthis study: H1, H7, H9, and H13 (which may be a mixture of two differentlines). The cells were maintained in the same media formulation onpoly-l-lysine coated plates. The cells proliferate when seriallypassaged.

A2B5-positive cells were induced to differentiate by the addition offorskolin. These cells have been assessed through different culturepassages, as shown in Table 3. TABLE 3 Phenotypic Features of MatureNeural Cells No. of Neuron-like Cells Staining Positively for: passagesafter Method of morphology β- A2B5 sort Maturation visible tubulin GFAPGalC A2B5 NCAM 1 PICNT + Fk yes 38 ± 9% 13 ± 7% 79 ± 3% 28 ± 6% 4 days 3PICNT + Fk yes +++ + +++++ ++ 2 days 7 +/− EF yes + + ++ +++ − +/− serumFactor abbreviations:C—ciliary neurotrophic factor (CNTF)F—basic fibroblast growth factor (bFGF)N—neurotrophin 3 (NT3)I—insulin-like growth factor (IGF-1)P—platelet-derived growth factor (PDGF)T—thyroid hormone T₃Ra—retinoic acidFk—Forskolin

Even though the cells were sorted for A2B5 expression, the populationdemonstrated the capacity to generate not only oligodendrocytes, andastrocytes, but also a large proportion of neurons. This is surprising:it was previously thought that A2B5 expressing cells were glialprecursors, and would give rise to oligodendrocytes, andastrocytes—while NCAM expressing cells were neuronal precursors, givingrise to mature neurons. This experiment demonstrates that pPS cells canbe differentiated into a cell population that proliferates repeatedly inculture, and is capable of generating neurons and glia.

Example 3 Differentiation to Mature Neurons

To generate terminally differentiated neurons, the first stage ofdifferentiation was induced by forming embryoid bodies in FBS mediumwith or without 10 μM retinoic acid (RA). After 4 days in suspension,embryoid bodies were plated onto fibronectin-coated plates in definedmedium supplemented with 10 ng/mL human EGF, 10 ng/mL human bFGF, 1ng/mL human PDGF-AA, and 1 ng/mL human IGF-1. The embryoid bodiesadhered to the plates, and cells began to migrate onto the plastic,forming a monolayer.

After 3 days, many cells with neuronal morphology were observed. Theneural precursors were identified as cells positive for BrdUincorporation, nestin staining, and the absence of lineage specificdifferentiation markers. Putative neuronal and glial progenitor cellswere identified as positive for polysialylated NCAM and A2B5. Forty oneto sixty percent of the cells expressed NCAM, and 20-66% expressed A2B5,as measured by flow cytometry. A subpopulation of the NCAM-positivecells was found to express β-tubulin III and MAP-2. There was noco-localization with glial markers such as GFAP or GaIC.

The A2B5 positive cells appeared to generate both neurons and glia. Asubpopulation of the A2B5 cells expressed β-tubulin III or MAP-2, and aseparate subpopulation expressed GFAP. Some of the cells with neuronalmorphology double-stained for both A2B5 and NCAM. Both the NCAM positiveand A2B5 positive populations contained far more neurons than glia.

The cell populations were further differentiated by replating the cellsin a medium containing none of the mitogens, but containing 10 ng/mLNeurotrophin-3 (NT-3) and 10 ng/mL brain-derived neurotrophic factor(BDNF). Neurons with extensive processes were seen after about 7 days.Cultures derived from embryoid bodies maintained in retinoic acid (RA)showed more MAP-2 positive cells (˜26%) than those maintained without RA(˜5%). GFAP positive cells were seen in patches. GaIC positive cellswere identified, but the cells were large and flat rather than havingcomplex processes.

A summary of cell types and markers expressed at different stages ofdifferentiation is provided in Table 4. TABLE 4 Phenotypic Markers(Immunocytochemistry) Undifferentiated hES colonies NCAM-positiveprogenitors A2B5 positive progenitors Tra-1-60 + Nestin subset Nestinsubset Tra-1-81 + A2B5 subset NCAM subset SSEA-4 + β-tubulin III subsetβ-tubulin III subset β-tubulin III + + Map-2 subset Map-2 subset Nestin− GFAP — GFAP rare Map-2 − GalC — GalC — Neurofilament (NF) − AFP — AFP— GFAP − muscle-specific actin — muscle-specific actin — GalC −α-fetoprotein − muscle-specific − actin NCAM − A2B5 − Neurons AstrocytesOligodendrocytes β-tubulin III + GFAP + GalC + MAP-2 + Neurofilament(NF) subset GABA subset tyrosine hydroxylase subset glutamate subsetglycine subset

The presence of neurotransmitters was also assessed. GABA-immunoreactivecells were identified that co-expressed β-tubulin III or MAP2, and hadmorphology characteristic of neuronal cells. Occasional GABA-positivecells were identified that did not co-express neuronal markers, but hadan astrocyte-like morphology. Neuronal cells were identified thatexpressed both tyrosine hydroxylase (TH) and MAP-2. Synapse formationwas identified by staining with synaptophysin antibody.

FIG. 2 shows TH staining in cultures differentiated from the H9 line ofhuman ES cells. Embryoid bodies were maintained in 10 μM retinoic acidfor 4 days, then plated onto fibronectin coated plates in EGF, basicFGF, PDGF and IGF for 3 days. They were next passaged onto laminin in N2medium supplemented with 10 ng/mL NT-3 and 10 ng/mL BDNF, and allowed todifferentiate further for 14 days. The differentiated cells were fixedwith 4% paraformaldehyde for 20 min at room temperature, and thendeveloped using antibody to TH, a marker for dopaminergic cells.

Example 4 Calcium Imaging

Standard fura-2 imaging of calcium flux was used to investigate thefunctional properties of the hES cell derived neurons. Neurotransmittersstudied included GABA, glutamate (E), glycine (G), elevated potassium(50 mM K⁺ instead of 5 mM K⁺), ascorbic acid (control), dopamine,acetylcholine (ACh) and norepinephrine. The solutions contained 0.5 mMof the neurotransmifter (except ATP at 10 μM) in rat Ringers (RR)solution: 140 mM NaCI, 3 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 10 mM HEPESbuffer, and 10 mM glucose. External solutions were set to pH 7.4 usingNaOH. Cells were perfused in the recording chamber at 1.2-1.8 mL/min,and solutions were applied by bath application using a 0.2 mL loopinjector located ˜0.2 mL upstream of the bath import. Transient rises incalcium were considered to be a response if the calcium levels roseabove 10% of the baseline value within 60 sec of application, andreturned to baseline within 1-2 min.

FIG. 3 shows the response of neural-restricted precursors to variousneurotransmitters. Panel A shows the ratio of emission data from singlecells on two different coverslips. Addition of the neurotransmitters isindicated above by labeled triangles.

Panel B shows the frequency of cells tested that responded to specificneurotransmitters. Panel C shows the combinations of neurotransmitterresponses observed. Of the 53 cells tested, 26 responded to GABA,acetylcholine, ATP and elevated potassium. Smaller subsets of thepopulation responded to other combinations of agonists. Only 2 of thecells failed to respond to any of the agonists applied.

Example 5 Electrophysiology

Standard whole-cell patch-clamp technique was conducted on the hES cellderived neurons, to record ionic currents generated in voltage-clampmode and the action potential generated in current-clamp mode. Theexternal bath solution was rat Ringers solution (Example 6). Theinternal solution was 75 mM potassium-aspartate, 50 mM KF, 15 mM NaCI,11 mM EGTA, and 10 mM HEPES buffer, set to pH 7.2 using KOH.

All 6 cells tested expressed sodium and potassium currents, and firedaction potentials. Passive membrane properties were determined withvoltage steps from −70 to −80 mV; and produced the following data:average capacitance (C_(m))=8.97±1.17 pF; membrane resistance(R_(m))=487.8±42.0 MΩ; access resistance (R_(a))=23.4±3.62 MΩ. Ioniccurrents were determined by holding the cells at −100 mV, and steppingto test voltages between −80 and 80 mV in 10 mV increments, producingthe following data: average sodium current I_(Na)=−531.8±136.4 pA;average potassium current I_(K=)441.7±113.1 pA;I_(Na)(density)=−57.7±7.78 pA/pF; I_(K)(density)=48.2±10.4 pA/pF.

FIG. 4 shows results from a typical experiment. Panel A shows sodium andpotassium currents observed in two cells depolarized to test potentialsbetween −80 and 80 mV from a holding potential of −100 mV. Panel B showsthe inward (Na⁺) and outward (K⁺) peak current-voltage relationshipsobserved. Sodium current activates between −30 and 0 mV, reaching a peakat −10 or 0 mV. Potassium current activates above −10 mV, becoming equalor larger in magnitude than the sodium current at voltages between 20and 40 mV. Panel C shows action potentials generated by the same cellsin response to depolarizing stimuli. Cell membranes were held atvoltages between −60 and −100 mV in −80 or −150 pA of current, anddepolarized for short durations

Example 6 Dopaminergic Cells Derived from Neural Progenitor Cells

Embryoid bodies were cultured in suspension with 10 μM retinoic acid for4 days, then plated into defined medium supplemented with EGF, bFGF,PDGF, and IGF-1 for 3-4 days. Cells were then separated by magnetic beadsorting or immunopanning into A2B5-positive or NCAM-positive enrichedpopulations.

The immuno-selected cells were maintained in defined medium supplementedwith 10 ng/mL NT-3 and 10 ng/mL BDNF. After 14 days, 25±4% of theNCAM-sorted cells were MAP-2 positive—of which 1.9±0.8% wereGABA-positive, and 3±1% were positive for tyrosine hydroxylase (TH): therate-limiting enzyme for dopamine synthesis, generally considered to berepresentative of dopamine-synthesizing cells.

In the cell population sorted for NCAM, the cells that were NCAM +ve didnot express glial markers, such as GFAP or GaIC. These data indicatethat a population comprising neuron restricted precursors can beisolated directly from hES cell cultures, essentially uncontaminatedwith glial precursors.

Cells sorted for A2B5, on the other hand, have the capacity to generateboth neurons and astrocytes. After the enrichment, the cells were placedinto defined media supplemented with NT-3 and BDNF and allowed todifferentiate for 14 days. Within the first 1-2 days after plating,cells in the A2B5 enriched population began to extend processes. Aftertwo weeks, cells took on the morphology of mature neurons, and 32±3% ofthe cells were MAP-2 positive. Importantly, 3±1% of the MAP-2 cells wereTH-positive, while only 0.6±0.3% were GABA immunoreactive. These dataindicate that a population of cells can be obtained from hES cells thatcomprise progenitors for both astrocytes and neurons, including thosethat synthesize dopamine.

Further elaboration of conditions for obtaining TH-expressing neuronswas conducted as follows. Embryoid bodies were generated from confluenthES cells of the H7 line at passage 32 by incubating in 1 mg/mLcollagenase (37° C., 5-20 min), scraping the dish, and placing the cellsinto non-adherent culture plates (Costar®). The resulting EBs werecultured in suspension in media containing FBS and 10 μM all-transretinoic acid. After four days, the aggregates were collected andallowed to settle in a centrifuge tube. The supernatant was thenaspirated, and the aggregates were plated onto poly L-lysine andfibronectin coated plates in proliferation medium (DMEM/F12 1:1supplemented with N2, half-strength B27, 10 ng/mL EGF (R & D Systems),10 ng/mL bFGF (Gibco), 1 ng/mL PDGF-M (R & D Systems), and 1 ng/mL IGF-1(R & D Systems).

The EBs were allowed to attach and proliferate for three days; thencollected by trypsinizing ˜1 min (Sigma) and plated at 1.5×10⁵cells/well onto poly I-lysine and laminin coated 4-well chamber slidesin proliferation medium for one day. The medium was then changed toNeural Basal medium supplemented with B27, and one of the followinggrowth cocktails:

-   -   10 ng/mL bFGF (Gibco), 10 ng/mL BDNF, and 10 ng/mL NT-3    -   10 ng/mL bFGF, 5000 ng/mL sonic hedgehog, and 100 ng/mL FGF8b    -   10 ng/mL bFGF alone        The cells were maintained in these conditions for 6 days, with        feeding every other day. On day 7, the medium was changed to        Neural Basal medium with B27, supplemented with one of the        following cocktails:    -   10 ng/mL BDNF, 10 ng/mL NT-3    -   1 μM cAMP, 200 μM ascorbic acid    -   1 μM cAMP, 200 μM ascorbic acid, 10 ng/mL BDNF, 10 ng/mL NT-3        The cultures were fed every other day until day 12 when they        were fixed and labeled with anti-TH or MAP-2 for        immunocytochemistry. Expression of the markers was quantified by        counting four fields in each of three wells using a 40×        objective lens.

Results are shown in Table 5. Initial culturing in bFGF, BDNF and NT-3yielded the highest proportion of TH positive cells. TABLE 5 Conditionsfor Producing Dopaminergic Neurons % of cells % MAP-2 that are cellsthat Culture conditions MAP-2 are TH days 1-6 days 6-12 positivepositive BDNF, NT-3, bFGF BDNF, NT-3 26% 5.5% BDNF, NT-3, bFGF cAMP, AA(ascorbic acid) 35% 4.0% BDNF, NT-3, bFGF cAMP, AA, BDNF, NT-3 25% 8.7%bFGF, FGF8, SHH BDNF, NT-3 37% 3.7% bFGF, FGF8, SHH cAMP, AA 34% 3.9%bFGF, FGF8, SHH cAMP, AA, BDNF, NT-3 21% 5.8% bFGF BDNF, NT-3 28% 3.5%bFGF cAMP, AA 26% 4.1% bFGF cAMP, AA, BDNF, NT-3 22% 5.7%

Example 7 Increased Proportion of Dopaminergic Cells by Culturing withErythropoietin

In a subsequent experiment, embryoid bodies were plated onto poly-lysinefibronectin coated wells, and cultured with 10 ng/mL EGF, 1 ng/mLPDGF-M, 10 ng/mL bFGF, and 1 ng/mL IGF-1. On the fourth day, the mixturewas supplemented with 5 U/mL EPO, 700 μM cAMP, or both. The cells werereplated and treated for 7 days with 10 ng/mL BDNF, 10 ng/mL NT-3, andoptionally EPO, cAMP, and 200 μM ascorbic acid. Results are shown inTable 6. The proportion of total cells in the culture that were MAP-2positive was abnormally low in this experiment. TABLE 6 Conditions forProducing Dopaminergic Neurons % MAP-2 cells Culture Conditions that aredays 1-3 days 4-5 days 6-12 TH positive (SD) EGF, bFGF, EGF, bFGF, BDNF,NT-3 20% (13%) PDGF, PDGF, IGF-1 IGF-1 (same) (same) BDNF, NT-3, 24%(3%) EPO, cAMP, AA (same) same plus EPO BDNF, NT-3, 31% (13%) EPO, cAMP,AA (same) same plus cAMP BDNF, NT-3, 47% (2%) EPO, cAMP, AA (same) sameplus EPO & BDNF, NT-3, 57% (7%) cAMP EPO, cAMP, AA

These data provide the first demonstration that adding cAMP and EPOduring derivation of the neural precursor cells increases the percentageof neurons ultimately obtained that expressed tyrosine hydroxylase.Studer et al. reported that proliferation and differentiation ofmesencephalic precursors in the presence of EPO or low partial pressuresof O₂ result in higher numbers of dopaminergic neurons (J. Neurosci.20:7377, 2000). EPO is thought to have a neuroprotective effect inhypoxic conditions, driving multipotent progenitors towards the neuronalpathway (Shingo et al., J. Neurosci. 21:9733, 2001). The effect may be aresult of cross-talk between Janus kinase-2 and nuclear factor kappaB(NF-κB), upregulation of Bcl-x(L) expression, or activation of AP-1(Jun/Fos) pathway. Regulating these pathways in pPS derived neural cellsby other means may mimic the effects of EPO.

Example 8 Direct Differentiation of hES Cells

In a parallel series of experiments, differentiation was initiated notby forming embryoid bodies, but by plating undifferentiated hES cellsdirectly onto a solid surface in the absence of feeder cells or otherfactors that inhibit differentiation.

Suspensions of rhesus and human ES cells were dissociated by triturationto clusters of ˜50-100 cells, and plated onto glass coverslips treatedwith poly-ornithine. The cells were maintained in serum containingmedium, or defined medium for 7-10 days before analysis. The cells werethen tested by immunoreactivity for β-tubulin III and MAP-2, which arecharacteristic of neurons, and glial fibrillary acidic protein (GFAP),which is characteristic of astrocytes.

Several different ES lines were differentiated into cells bearingmarkers for neurons and astrocytes, using either the aggregate or directdifferentiation technique. In cultures derived from rhesus ES cells,percentage of aggregates that contained neurons ranged from 49% to 93%.In cultures derived from human ES cells, the percentage of aggregatescontaining neurons ranged from 60% to 80%. Double labeling for GABA andβ-tubulin indicated that a sub-population of the neurons express theinhibitory neurotransmifter GABA. Astrocytes and oligodendrocytes wereidentified with GFAP immune reactivity and GaIC immune reactivity,respectively. Therefore, the human and rhesus ES cells have the capacityto form all three major cell phenotypes in the central nervous system.

The effect of several members of the neurotrophin growth factor familywas examined. hES cells were differentiated by harvesting withcollagenase, dissociating, and reseeding onto poly-ornithine coatedcover slips. The cells were plated into DMEM/F12+N2+10% FBS overnight.The following day, the serum was removed from the medium and replacedwith 10 ng/mL human bFGF and the growth factor being tested. After 24hours, bFGF was removed from the medium. These cultures were fed everyother day. They were fixed after 7 days of differentiation andimmunostained for analysis. The number of neurons was evaluated bycounting cells positive for β-tubulin. Cultures maintained in thepresence of 10 ng/mL brain derived neurotrophic factor (BDNF) formedapproximately 3-fold more neurons than the control cultures. Culturesmaintained in neurotrophin-3 (1 ng/mL) formed approximately 2-fold moreneurons than control cultures.

Example 9 Direct Differentiation of hES Cells to Dopaminergic Neurons

This study evaluated various paradigms for differentiating human EScells into neurons without the formation of embryoid bodies.

A strategy was developed in which the test factors were placed intogroups based on homology and/or functional redundancy (Table 3).Grouping factors increases the likelihood that an activity associatedwithin that group will be elicited on the ES cell population. Thehypothesis is that certain factors within the mixture will initiate adifferentiation cascade. As differentiation proceeds, and the receptorexpression profile of the cells change, they will become responsive toother factors in the mixture.

Providing a complex mixture of factors continuously over the treatmentperiod avoids the need to define exactly how and when the responsivenessof the cells changes. When a mixture is identified that elicits thedesired differentiation process, it can be systematically simplified toachieve a minimal optimal mixture. After further testing, minimaltreatment may ultimately comprise one, two, three, or more of thefactors listed, used either simultaneously or in sequence according tothe empirically determined protocol. TABLE 7 Test Factor Groups Group 1Group 2 Group 3 Neurotrophins Mitogens Stem Cell Factors 30 ng/mL NGF 30ng/mL EGF  8 ng/mL LIF 30 ng/mL NT-3 30 ng/mL FGF-2 (basic FGF)  3 ng/mLIL-6 30 ng/mL NT-4 37 ng/mL FGF-8b  3 ng/mL IL-11 30 ng/mL BDNF 30 ng/mLIGF-I  3 ng/mL SCF 30 ng/mL PDGF-AA 30 ng/mL CNTF Group 4 Group 5Differentiation Factors TGF-β Group 6 TGF-β Superfamily SuperfamilyAntagonists Differentiation Factor 30 ng/mL BMP-2 150 ng/mL Noggin 37ng/mL SHH 37 ng/mL GDF-5  30 ng/mL Follistatin  3 ng/mL GDNF 30 ng/mLNeurturin Group 7 Group 8 Group 9 Neurotrophic Factor DifferentiationFactor Survival Factor/Antioxidant 37 ng/mL Midkine 17 μM Retinoic Acid166 μM Ascorbic Acid Group 10 Differentiation Factor/ Group 11Neurotransmitter Survival Factor 10 μM Dopamine 100 μM Dibutyryl cAMP

The experiment was conducted as follows. Monolayer cultures of a humanES cell line were harvested by incubating in Collagenase IV for 5-10min, and then scraping the cells from the plate. The cells weredissociated by trituration and plated at subconfluence onto 96 welltissue culture plates pretreated with growth factor-reduced Matrigel® inKnockout DMEM medium (Gibco BRL) with Knockout Serum Replacement (GibcoBRL) conditioned 24 h by mouse embryonic feeder cells One day afterplating, the medium was replaced with Neurobasal (NB) Medium (Gibco BRL)supplemented with 0.5 mM glutamine, B27 supplement (Gibco BRL) andgroups of test factors as described below. The cells were fed daily withfresh Neurobasal Medium containing glutamine, B27, and test factors for11 days.

After 11 days, the cells were harvested by incubation in trypsin for5-10 min, replated at a 1:6 dilution onto 96 well tissue culture platespretreated with laminin, and fed daily with fresh Neurobasal Mediumcontaining glutamine, B27 and test factors for an additional 5 days.Cells were fixed for 20 min in 4% paraformaldehyde, and stained withantibodies to the early neuronal marker, β-Tubulin-III, the lateneuronal marker, MAP-2, and tyrosine hydroxylase, an enzyme associatedwith dopaminergic neurons. Cell nuclei were labeled with DAPI, andquantified by visual inspection. Results are shown in Table 8. TABLE 8Direct Differentiation of hES Cells to Neurons Tyrosine βTubulin-IIIMAP-2 Hydroxylase positive positive positive Test Compound Groups Cells/Cells/ Cells/ Included in Cell Culture Well % Total Well Well % TotalControl 102 —  2  1 — Treatment 1, 2, 3, 4, 6, 7, 8, 9, 10, 11  0 0  0 0 — A: Treatment 1, 2, 3, 5, 6, 7, 8, 9, 10, 11 362  6% 132 14 0.2% B:Treatment 1, 2, 4, 6, 7, 8, 9, 10, 11 — — — — — C: Treatment 1, 2, 5, 6,7, 8, 9, 10, 11 378 11% 162 16 0.5% D: Treatment 1, 3, 4, 6, 7, 8, 9,10, 11  6 —  2  4 — E: Treatment 1, 3, 5, 6, 7, 8, 9, 10, 11 282 12%  92 4 0.2% F: Treatment 1, 4, 6, 7, 8, 9, 10, 11  17 —  0  2 — G:— = not determined

In another experiment, cells were cultured in Neurobasal Mediumsupplemented with glutamine, B27 and groups of test factors as before,harvested with trypsin at 8 days, and replated for 5 days. Results areshown in Table 9. TABLE 9 Direct Differentiation of hES Cells to NeuronsPercent of MAP-2 Tyrosine positive βTubulin-III MAP-2 Hydroxylase cellsalso Test Compound Groups positive positive positive positive forIncluded in Cell Culture Cells/Well Cells/Well Cells/Well TH Control 4 40 Treatment 1, 2, 3, 4, 6, 7, 8, 9, 10, 11 12 8 3 A: Treatment 1, 2, 3,5, 6, 7, 8, 9, 10, 11 268 12 4 B: Treatment 1, 2, 4, 6, 7, 8, 9, 10, 1112 0 0 C: Treatment 1, 2, 5, 6, 7, 8, 9, 10, 11 372 48 7 15% D:Treatment 1, 3, 4, 6, 7, 8, 9, 10, 11 0 0 0 E: Treatment 1, 3, 5, 6, 7,8, 9, 10, 11 196 56 0 F: Treatment 1, 4, 6, 7, 8, 9, 10, 11 16 0 9 G:

Several treatment paradigms induced the direct differentiation ofneurons. Treatments that included Group 5 factors (noggin andfollistatin) were the most effective.

FIG. 5 shows exemplary fields of differentiated cells obtained usingTreatment B, Treatment D, and Treatment F, and stained forβ-tubulin-III. About 5-12% of the cells are neurons, based on morphologyand β-tubulin-III staining. About ⅓ of these are mature neurons, basedon MAP-2 staining. About 2-5% of total neurons (5-15% of MAP-2 positiveneurons) also stained for tyrosine hydroxylase, which is consistent witha dopaminergic phenotype.

Subsequent experiments have been conducted to further elucidate theeffect of certain factor cocktails and the kinetics of differentiation.

FIG. 6(A) shows the results of an experiment in which the TGF-βsuperfamily antagonists noggin and follistatin were used for varyingtime periods. Subconfluent hES cells of the H7 line were treated for 15days with Treatment D, except that cAMP concentration was 700 μg. Theresults indicate that noggin and follistatin both contribute to neurondifferentiation, and work synergistically. Noggin is apparentlyimportant at about the 1 week point (days 5 to 8), while follistatin isimportant at around the 2 week point (days 13 to 15), maximizingproduction of mature neurons rather than small neurites.

FIG. 6(B) shows the time course of neuronal induction using thetreatment mixtures in Table 8 containing TGF-β superfamily antagonists.FIG. 6(C) further illustrates the effects of noggin and follistatin indirect differentiation. hES cells represented by the first bar weretreated with the factors of Groups 1, 4, 6, 7, 9, 10, and 11 (Table 7),with 700 μM cAMP, 5 U/mL EPO, plus 30 ng/mL FGF-8 (Group 2). Virtuallyno β-tubulin positive neurons were formed in the absence of noggin orfollistatin. However, noggin and follistatin alone or in combinationwith retinoic acid directly induced hES cells through the first steps ofneuronal differentiation. It is hypothesized that initialnoggin/follistatin induction generates a neural progenitor cell, whichsubsequently can be induced to form neurons by the addition of otherfactors.

FIG. 6(D) shows the benefit of omitting retinoic acid (RA) from themixture where dopaminergic neurons are desired. Cells weredifferentiated according to treatment F as previously (left 2 bars) oromitting retinoic acid (right 2 bars). Including retinoic acid increasedthe total percentage of β-tubulin positive neurons somewhat, butdecreased the proportion of those neurons staining positively fortyrosine hydroxylase.

Example 10 Proliferative Regeneration of Neural Precursors by SerialPassaging

The neural progenitors of this invention can be passaged and expanded inculture, demonstrating some of their unique and beneficial properties.

In an exemplary experiment, human embryonic stem cells were harvestedand placed into suspension culture to form embryoid bodies in knockoutDMEM containing 20% FBS plus 10 μM retinoic acid. After 4 days, theembryoid bodies were plated onto poly-L-lysine/fibronectin-coated platesin DMEM/F12 medium supplemented with N2 supplement, B27 supplement athalf the usual amount, 10 ng/mL human EGF, 10 ng/mL human bFGF, 1 ng/mLhuman PDGF-AA, and 1 ng/mL human IGF-1.

The cells were cultured for 3 days, and harvested by brieftrypsinization as follows. Half a mL 0.5% Trypsin in 0.53 mM EDTA (Gibco#25300-054) was layered into each well of a 6-well plate, thenimmediately removed from the plate. After waiting 15 seconds (roomtemperature), neurobasal medium plus B27 supplement was placed in thewells, and then removed and centrifuged to recover the released cells(between 1 and 10% of the cells).

Six-well plates were coated with 1 mL/well of 15 μg/mL poly-L-lysine(Sigma #P1274), followed by 1 mL/well of 20 μg/mL human placentallaminin (Gibco #23017-015) overnight. The cell pellet from thedifferential trypsinization was resuspended in neurobasal mediumcontaining B27 supplement, 10 ng/mL NT-3, and 10 ng/mL BDN F, and platedonto the coated wells at 500,000 to 750,000 cells per well.

After 5 days, the cells were recovered by complete trypsinization,counted, and replated at 100,000 to 150,000 cells per well in newpoly-lysine/laminin coated wells in the presence of various factorcocktails. Concentrations used were as follows: 10 ng/mL NT-3, 10 ng/mLBDNF, 10 ng/mL human EGF, 10 ng/mL human bFGF, or 10 ng/mL LIF, invarious combinations. The cells were fed with a half exchange of mediumthree times per week. Every 7 days, the cells were trypsinized, counted,and passaged again in fresh medium containing the same factors.

FIG. 7(A) shows the growth curves from this experiment. Cells passagedin BDNF and NT-3 alone stop growing after ˜1 week, predominantlydifferentiating into neurons. However, adding EGF and bFGF to the mediumallowed the cells to continue proliferating in the precursor form. Themarker profile of these cells is shown in Table 10. TABLE 10 Phenotypeof Neural Progenitors Markers Tyrosine Cocktail Passage Nestin PS-NCAMA2B5 β-tubulin III GFAP MAP2 Hydroxylase NT-3, BDNF, p4 +++ +++ ++ + + −− EGF, bFGF, LIF p8 +++ +++ + + + − − NT-3, BDNF, p4 +++ +++ + + + − −EGF, bFGF p8 +++ +++ − + + − − EGF, bFGF, LIF p4 ++ ++ + + + − − p8 ++++ − + − − − EGF, bFGF p4 ++ ++ − − − − − p8 + + − − − − −Thus, cells passaged in a combination of BDNF, NT-3, EGF, and bFGFabundantly expressed the neural progenitor markers Nestin and NCAM.

FIG. 7(B) shows results obtained when these cells were induced toterminally differentiate in BDNF and NT-3 alone. The cells passaged in acombination of BDNF, NT-3, EGF and bFGF produced more neurons uponterminal differentiation, consistent with the higher proportion ofneural precursors before differentiation.

FIG. 8(A) shows the proportion of cells staining positively for tyrosinehydroxylase. Again, the combination of BDNF, NT-3, EGF and bFGF providedoptimal yield amongst the combinations tested.

FIG. 8(B) shows that even more TH-positive neurons can be generated byinducing terminal differentiation not by BDNF and NT-3 alone, but alsoincluding additional factors such as NT-4, nerve growth factor, ascorbicacid, cAMP and dopamine (at the concentrations shown in Table 7). Up to5% of the total cell number in the population displayed the phenotype ofdopaminergic markers.

Neural progenitors from the H7 hES cell line were frozen down at passage10 in neural basal medium containing B27 supplement, 30% serumreplacement, and 10% DMSO (5×10⁵ cells per freezing vial). The cellswere thawed about 6.5 months later. The thawed cells had many of thesame characteristics that they did before freezing: 60-80% β-tubulin andMAP-2 positive, ˜5% positive for tyrosine hydroxylase.

In a related experiment, cells were grown and passaged as clustersrather than on a culture substrate. Neural progenitors were harvestedusing trypsin from a 6 well plate when nearly confluent (˜3 or 4×10⁵cells per well). They were then seeded at ˜2.5×10⁵ cells per well innonadherent wells, and cultured in 2 mL neural basal medium containingB27 supplement, 10 ng/mL BDNF, 10 ng/mL NT-3, 10 ng/mL EGF, and 10 ng/mLbFGF. The cells were fed the following day by exchanging half themedium, and cultured for a following 4 days. They were thendifferentiated in medium containing 10 ng/mL BDNF and 10 ng/mL NT-3 butno mitogens.

Adaptations of the invention described in this disclosure are a matterof routine optimization, and can be done without departing from thespirit of the invention, or the scope of the claims below.

1. A system for generating human neuronal cells, comprising: a firstcell population comprising undifferentiated cells from a line of humanembryonic stem (hES) cells; and a differentiated cell populationcultured in vitro, wherein at least ˜30% of MAP-2 positive cells havethe characteristic that they are progeny of the hES cells, and expresstyrosine hydroxylase.
 2. A system for generating human neuronal cells,comprising: a first cell population comprising undifferentiated cellsfrom a line of human embryonic stem (hES) cells; and a differentiatedcell population cultured in vitro, wherein at least ˜5% of all the cellsin the population have the characteristic that they are progeny of thehES cells, and express tyrosine hydroxylase.
 3. A system for generatinghuman neuronal cells, comprising: a first cell population comprisingundifferentiated cells from a line of human embryonic stem (hES) cells;and a neuronal precursor cell population cultured in vitro, in which atleast ˜60% of the cells are progeny of the hES cells; express A2B5,polysialylated NCAM, or Nestin; and which upon culturing for 7 days withadded neurotrophin 3 (NT-3), brain-derived neurotrophic factor (BDNF),neurotrophin 4 (NT-4) and nerve growth factor (NGF), but no addedmitogens, generates a cell population in which at least ˜30% of MAP-2positive cells express tyrosine hydroxylase.
 4. A system for generatinghuman neuronal cells, comprising: a first cell population comprisingundifferentiated cells from a line of human embryonic stem (hES) cells;and a neuronal precursor cell population cultured in vitro, in which atleast ˜60% of the cells are progeny of the hES cells; express A2B5,polysialylated NCAM, or Nestin; and which upon culturing for 7 days withadded neurotrophin 3 (NT-3), brain-derived neurotrophic factor (BDNF),neurotrophin 4 (NT-4) and nerve growth factor (NGF), but no addedmitogens, generates a cell population in which at least ˜5% of all thecells in the population express tyrosine hydroxylase
 5. The system ofclaim 3, wherein the neuronal precursor cell pupation is capable of atleast 20 population doublings in culture, and which after 20 doublingsmaintains an ability to form differentiated cell populations accordingto claim 1 or 2 upon culturing with NT-3, BDNF, NT-4 and NGF, but noadded mitogens.
 6. The system of claim 3, wherein the neuronal precursorcell population provides clinical improvement in a nigrostriatal lesionanimal model of Parkinson's disease.
 7. The system of claim 3, whereinat least ˜38% hES derived neural cells express β-tubulin III.
 8. Thesystem of claim 3, produced by: a) obtaining a line of hES cells; b)culturing some of the hES cells in a medium containing one or moreneurotrophins and one or more mitogens, and c) harvesting from theculture a cell population in which at least ˜60% of the cells expressA2B5, polysialylated NCAM, or Nestin; which is capable of at least 20doublings in culture, and which after 20 doublings maintains an abilityto be differentiated into a population comprising at least 20% MAP-2positive cells.
 9. The system of claim 8, wherein the added mitogen(s)include a mitogen selected from epidermal growth factor (EGF), basicfibroblast growth factor (bFGF), platelet-derived growth factor (PDGF),insulin-like growth factor 1 (IGF-1), and erythropoietin (EPO).
 10. Thesystem of claim 8, wherein the added neurotrophins include neurotrophin3 (NT-3) or brain-derived neurotrophic factor (BDNF).
 11. The system ofclaim 3, produced by: a) obtaining a line of hES cells; a) culturingsome of the hES cells in a medium containing one or more added TGF-βsuperfamily antagonists, and b) harvesting from the culture a neuralcell population in which at least 50% of the cells express eitherpolysialylated NCAM or β-tubulin III.
 12. The system of claim 11,wherein the neural cell population was produced by culturing the hEScells in a medium containing both noggin and follistatin.
 13. The systemof claim 8, wherein the neural cell population was produced by passagingthe cells at least 6 times in a medium comprising an added neurotrophinand an added mitogen.
 14. The system of claim 1, produced by: a)obtaining a line of hES cells; b) differentiating some of the hES cellsto produce a neuronal precursor cell population in which at least ˜60%of the cells express A2B5, polysialylated NCAM, or Nestin, and then c)culturing the neuronal precursor cells in a medium containing one ormore factors selected from neurotrophins, cAMP, and ascorbic acid in theabsence of added mitogens, so as to generate a cell population in whichat least ˜30% of MAP-2 positive cells express tyrosine hydroxylase